Thyroid nodule disease is a common clinical problem, found in 4-7% of the living adult population in North America. The occurrence of thyroid nodules increases with age; autopsies reveal the presence of thyroid nodules in 50% of the population. It is estimated that, at 80 years old, 90% of the population will have at least one thyroid nodule. However, the vast majority of solitary thyroid nodules are benign in nature, and would require no further treatment if a correct diagnosis could be obtained without surgery.
A number of techniques can be used to diagnose thyroid conditions, including radioactive thyroid scans, ultrasound, thyroid hormone level and thyroglobulin measurements, and fine needle aspiration biopsy (FNAB). Thyroid scans do not effectively distinguish benign and malignant conditions, however, and are typically used in conjunction with other techniques. Similarly, ultrasound may provide information suggestive of either benign or malignant conditions, but cannot definitively diagnose thyroid status. Measurements of thyroid hormone level and thyroglobulin can be informative, but are nondiagnostic by themselves.
Thyroid FNAB is the only non-surgical method which can by itself differentiate malignant and benign nodules. More than 300,000 fine needle aspiration biopsies (FNAB) of the thyroid are performed annually in the US and evaluated using cytology. The primary purpose of FNAB is to distinguish thyroid nodules that require immediate surgical intervention (e.g., total thyroidectomy in the case of a diagnosis of malignant disease) from nodules that can be treated effectively with less aggressive clinical approaches.
In FNAB, samples of thyroid cells are obtained by inserting a needle into the thyroid and aspirating cells into a syringe. Usually, 2 to 4 aspirations are made from different sites in each nodule. The cells are mounted on a slide (for each aspiration, 2 to 4 slides are prepared), stained, and examined. The sample is then classified as nondiagnostic (indeterminate), benign, suspicious or malignant. Most samples are categorized as benign.
FNAB can be used to successfully diagnose papillary carcinoma, medullary carcinoma, anaplastic carcinoma, thyroid lymphoma and metastases to the thyroid from other sites. Papillary carcinoma accounts for ˜60-70% and the follicular variant of papillary carcinoma accounts for ˜6% of thyroid cancers. These well differentiated thyroid cancers are usually curable, but they must be found first.
Especially problematic are cases considered ‘suspicious’, ‘inadequate’ or ‘indeterminate’ by cytological diagnosis of FNAB samples. These patients are invariably triaged by invasive surgery, which has a significant morbidity. Overtreatment with total-thyroidectomy frequently occurs as a result; it is estimated that less than 25% of patients with such diagnoses in fact have cancer that warrants removal of the thyroid gland. Approximately 5-10% of samples are classified as nondiagnostic by FNAB. In those cases, FNAB can be repeated; however, only half of repeat biopsies yield a diagnostic result. For the remaining patients, further testing and surgery may be required. Due to the fear of cancer, invasive surgery is chosen, but in most cases is unnecessary. Approximately 10-20% of samples are classified as suspicious by FNAB. Of these, approximately 25% will ultimately prove to be malignant after surgery, typically exhibiting follicular or Hurthle cell cancers, which cannot be diagnosed by FNAB. Follicular carcinoma, which accounts for ˜12-15% of all thyroid cancers and the less prevalent Hurthle cell carcinoma cannot be distinguished cytologically from benign follicular or Hurthle cell adenomas. Therefore, most patients with suspicious biopsies are typically subjected to surgery, when in fact ˜75% of these patients do not have malignant disease.
A contributing factor to the difficulties with current FNAB cytology-based diagnoses is the variability between different pathologists and cytopathologists in diagnostic agreement between cytological analysis and final histological review, ranging from 40%-90%. The overall accuracy of diagnoses using only FNAB ranges from 60% to >90%, and is dependent on the expertise of the cytologist and whether or not ‘suspicious’ or ‘indeterminate’ diagnostic categories are included in the reported accuracy of the study. When factoring the cytology diagnostic categories of ‘suspicious’ or ‘indeterminate’, the literature shows that the overall specificity of FNAB cytology for diagnosis of malignant disease decreases dramatically to <60% with false-positive rates of .about.40%. Patients with malignant thyroid disease are invariably treated by total removal of the tumor and all of the thyroid gland followed by radioactive iodine treatment, whereas benign thyroid disease can be treated less aggressively with a near-total thyroidectomy, partial thyroidectomy (e.g., ‘lobectomy’) or a watchful-waiting approach (e.g., observation without surgical intervention). As FNAB and cytology cannot reliably distinguish malignant from benign disease in cases with ‘suspicious’ cytological findings, such as occurs in the case of follicular and Hurthle cell lesions, these patients are typically all treated as if they were diagnosed with malignant disease (i.e., with aggressive surgery). Since only a small fraction of these patients in fact have malignant disease, over-treatment of thyroid nodule disease patients occurs frequently, with significant consequences for patients. As such, many unnecessary thyroidectomies are therefore performed in patients with what ultimately proves to be benign or non-neoplastic thyroid nodule disease when an FNAB sample is deemed as ‘suspicious’ or ‘indeterminate.’ These deficiencies negatively impact patient outcomes, long-term well-being and healthcare efficiencies.
Use of molecular analyses has the potential to increase the sensitivity, specificity and/or overall accuracy of thyroid diagnoses as compared to FNAB cytology alone. In the pre-operative setting, such a result would likely reduce the number of unnecessary surgeries for patients without malignant disease and avoid inadvertent undertreatment of highly curable thyroid cancers resulting from misdiagnoses. In addition, an accurate molecular based diagnosis as an adjunct assay to established pathological review diagnosis of thyroidectomy specimens in the post-operative setting could be beneficial by increasing the confidence of pathologists in establishing a definitive diagnosis for cancer that would likely influence the course of treatment and management of definitive malignant disease. However, prior attempts at using gene expression profiling to develop diagnostic gene expression signatures and identify mRNA biomarkers useful for the differential diagnosis of thyroid nodule disease have not yet yielded new clinical tools to improve the diagnosis of malignant from benign thyroid nodule disease from clinical specimens. Most of these efforts and those of protein immunohistochemistry studies focused on the protein-encoding genome. However, the transcriptome is inherently more complex than this, given that <2% of the genome encodes for protein and recent studies that have shown that more than 90% of the genome undergoes transcription yielding millions of non-coding RNA transcripts that serve regulatory roles over the protein-encoding transcriptome. So, gene-level analysis may provide only a rough estimate of diagnosis as it cannot capture the full differences between the genomes of malignant and benign thyroid nodule disease (e.g., alternative gene splicing, non-coding and functional RNA expression). Recent efforts to validate a 3-gene signature for diagnosis of thyroid nodule disease FNAB with a QRT-PCR approach report a low diagnostic accuracy in a large validation study (see Sibru et al., citation #14). Other prior attempts using gene-biased microarrays showed similar performance characteristics with low diagnostic accuracy for gene-based signatures (see Jiang et al., US 2007/0037186 A1). For example, Jiang et al., (US 2007/0037186 A1) disclosed a 4-gene QRT-PCR panel with a sensitivity of 92% but a specificity of just 61%. As a result, diagnoses using these provide results little better than FNAB cytology. In addition, other prior attempts utilize samples which are generally not available in the clinical setting. In particular, in the majority of clinical settings fresh tissue is unavailable. Formalin fixation is an essential part of the routine processing of tissue samples because this fixative best preserves the architecture of the tissue and cellular morphology, allowing pathologists enough definition to ascertain a diagnosis. Fresh or frozen unfixed tissue is suboptimal for viewing key details that pathologists use to differentially diagnose disease (e.g., benign vs cancer). For example, US 2008/0145841 and WO2006/127537 describes a thyroid fine needle aspiration molecular assay using fresh frozen samples. WO 2006/127537 showed a best result of 92% specificity and 76% sensitivity (see Table 12, page 98) and US 2008/0145841 showed an accuracy of 87.1% (see para [0127]).
Another possible reason why previous efforts aimed to developing molecular based classification schemes for thyroid nodule disease have not led to routine clinical assays relates to the technical feasibility of administering a molecular test. Typically, thyroid nodule fine-needle aspirate biopsies provide only a small amount of cells and therefore only minute yields of extractable nucleic acids or proteins that may be insufficient for standard molecular assays. In addition, many FNABs are further processed to prepare cell blocks or cell pellets made by centrifuging a fine-needle aspirate, followed by fixation similar to an FFPE block. After surgical resection, standard pathology practices require detailed post-operative evaluation of thyroidectomy specimens; this is especially important to establish a definitive diagnosis of cancer in cases where the FNAB cytology results were only ‘suspicious’ or indeterminate for the presence of cancer. Both of these procedures involve formalin-fixation and paraffin embedding, as this procedure best preserves the morphology and definition of the cells (in comparison to fresh or frozen preparates) favored by pathologists for microscopic evaluation but problematic for many nucleic acid molecular assays due to fragmentation of nucleic acids by formalin-fixation and paraffin embedding. Therefore, small amounts of sample and the use of fixatives are two additional technical impediments that must be overcome in order to apply molecular analyses of nucleic acids in routine clinical settings.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.